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    • There are multiple ways whereby oxidative stress may promote

    2018-10-26

    There are multiple ways whereby oxidative stress may promote FOXO activation. For example, FOXO proteins can directly sense ROS through the ability of ROS to induce disulfide bridges between cysteine residues on the FOXO protein, resulting in FOXO activation and transcription of ROS-removal diazoxide (Dansen, 2011). However, while this would explain the noted increase in FoxO1 and FoxO3a nuclear localization, it does not explain the Glc-dependent increase in FoxO1 and FoxO3a mRNA expression that was also observed. Nonetheless, the Glc-mediated FoxO1 and FoxO3a mRNA induction could potentially be due to a previously described relationship between ROS, phosphorylated JNK, and FoxO transcription (Essers et al., 2004) that goes beyond the protein level investigated here. Furthermore, treatment with antioxidants in this study reversed the Glc-dependent increase in FOXO3a nuclear localization and Sod2 and Cat expression, suggesting that the generation of excess ROS is indeed upstream of FOXO3a target expression. Interestingly, treatment with antioxidants led to a global increase in CAT activity that was unexpected, but could be due to the alleviation of TCF7L1-regulated repression following antioxidant treatment, leading to an increase in gene expression (Solberg et al., 2012). An unanswered question remains the identity of the kinase responsible for nuclear CTNNB1 accumulation. Our data suggested that it is not AKT. Treatment with the AKT inhibitor 124005 in physiological Glc conditions resulted in an increase in nuclear FOXO3a, but did not affect CTNNB1 nuclear localization, demonstrating that nuclear CTNNB1 localization in hyperglycemia is independent of AKT inhibition. These data are inconsistent with previous findings, as AKT has been shown to promote an increase in CTNNB1 transcriptional activity through the phosphorylation of S552 (Fang et al., 2007) and also to inhibit GSK3β, which normally phosphorylates CTNNB1 on S33/37/T41, tagging it for degradation (Shiojima and Walsh, 2006; Srivastava and Pandey, 1998). One possible explanation for this could be the existence of different pools of CTNNB1 that are regulated independently of one another (Mbom et al., 2013), or perhaps that CTNNB1 is regulated differently in stem cells than in other cell types. Our findings also raise questions regarding current ESC culture techniques. Routinely used media contain 25 mM Glc, and it is often observed that there is a tremendous amount of spontaneous differentiation and that ESCs behave differently from passage to passage. In fact, one study demonstrated that the derivation and culture of ESCs in physiological Glc reduced oxidative stress and improved proliferative capacity (Wang et al., 2006) while a different study confirmed that exposure to oxidative stress promoted cell-cycle arrest and senescence (Guo et al., 2010). Our contribution here is to have provided a molecular consequence of oxidative stress on ESCs, mediated through FOXO3a activation and AKT inhibition. Recent studies have demonstrated that FOXOs are important in ESC identity, with FOXO1 being a positive regulator in the maintenance of pluripotency, while the role of FOXO3a remains elusive (Zhang et al., 2011) and stimulation of AKT in ESCs promotes proliferation (Heo and Han, 2006). Our results are consistent with these reports, demonstrating that hyperglycemia results in an increase in oxidative stress that leads to changes in the proliferative capacity of pluripotent cells mediated partly through the activation of FOXO3a downstream of AKT and JNK regulation.
    Experimental Procedures
    Acknowledgments The authors thank Dr. Xiao-Fan Wang (Duke University Medical Center, Durham, NC) for generously providing the p21 luciferase reporter constructs, Dr. Boudewijn Burgering for the pSuperior-FoxO1/3 plasmid, and Dr. Irving Weissman (Stanford University) for the LEF/TCF-GFP ESCs. We further express gratitude to Susann Horvat, Aashima Singhal, and Beatrice Kuske for excellent technical assistance. This study was supported by start-up funds from the University of California Riverside.